Uterine milk protein, a novel activin-binding protein, is present in ovineallantoic fluid

Activins are pluripotent growth factors that have recently been shown to be present in placental and fetal membrane preparations. Our previous studies have identified and purified activin A from ovine amniotic and allantoic f luids. In this study, ligand blots of side fractions from the isolation of activin A from allantoic fluid suggested the presence of activin-binding pr oteins other than follistatin. Further purification of one of these fractio ns involved two sequential reverse phase HPLC steps and a Superose 12HR fra ctionation. SDS-PAGE revealed a single protein band of 55 kDa, which was id entified by NH2-terminal sequencing as ovine uterine milk protein (UTMP), a member of the serine protease inhibitor (serpin) superfamily of proteins. Further binding studies, using ligand blot techniques and Superose 12HR fra ctionation in the presence of [I-125]activin, demonstrated UTMP to be an ac tivin-binding protein with a lower affinity for activin than that of follis tatin. A study of the specific binding behavior of UTMP to activin, using s urface plasmon resonance, revealed an apparent equilibrium dissociation con stant (K-d) of 49 +/- 25 nM, compared with the follistatin-activin K-d of 3 79 +/- 51 pM. Similar to another activin-binding protein, alpha(2)-macroglo bulin, UTMP was unable to neutralize the bioactivity of activin in a bioass ay based on the capacity of activin to inhibit the proliferation of an MPC- 11 plasmacytoma cell line. The high concentrations of this protein in uteri ne fluid during pregnancy and its ability to bind activin suggest that UTMP may act as a low affinity, high capacity binding protein to sequester acti vin in the local uterine environment.